Inorganic phosphate ion (PO.sub.4.sup.3-, or P.sub.i) is a critical component in many biological systems, and so a variety of assays have been developed for the detection and/or quantification of inorganic phosphate. While previous methods have utilized coupled enzyme reactions, none of the previously described methods for detecting or quantitating inorganic phosphate utilized the fluorogenic peroxidase substrates of the present invention, and did not offer the sensitivity and accuracy of the methods of the instant invention.
The instant invention describes highly sensitive methods for the enzymatic determination of inorganic phosphate, including the consumption or production of phosphate, by coupling the system of interest to a probe for hydrogen peroxide. The system of interest is coupled enzymatically to a reaction producing hydrogen peroxide, which is coupled to a peroxidase enzyme reaction in the presence of a chromogenic or fluorogenic substrate. Unlike the methods previously used for detecting and monitoring phosphate, the methods of the instant invention are highly sensitive, may be utilized at wavelengths that are more compatible with biological samples, may be carried out at physiological pH, and permit a continuous assay. Given its high sensitivity and one-step procedure, the method of the invention is a valuable tool for measuring a variety of phosphate-dependent enzymes in biological samples, particularly as a microplate-based assay for high-throughput applications.